Cloth-based hybridization array system for the detection of Clostridium botulinum type A, B, E, and F neurotoxin genes.

A simple cloth-based hybridization array system was developed for the characterization of Clostridium botulinum isolates based on the botulinum neurotoxin serotype. Bacterial isolates were subjected to a multiplex PCR incorporating digoxigenin-dUTP and primers targeting the four botulinum neurotoxin gene serotypes (A, B, E, and F) predominantly involved in human illness, followed by hybridization of the amplicons with an array of toxin gene-specific oligonucleotide probes immobilized on polyester cloth and subsequent immunoenzymatic assay of the bound digoxigenin label. This system provided sensitive and specific detection of the different botulinum neurotoxin gene markers in a variety of C. botulinum strains, exhibiting the expected patterns of reactivity with a panel of target and nontarget organisms.

Extraction of Salmonella antigens in non-sedimentable forms for enzyme immunoassay.

Heating Salmonella typhimurium in ethylenediaminetetraacetate was found to dissociate its antigens into forms that are non-sedimentable at 10,000 x g. The treatment caused a marked increase in the rate of immunoreaction and the sensitivity in an enzyme immunoassay for the detection of Salmonella antigens. The method permits the extraction of Salmonella antigens from solid-rich samples and the preparation of solid-free samples by means of centrifugation. When the level of the antigens in the supernatant was too low for the immunoassay, the antigens were readily concentrated by passing a large volume of the supernatant through a macroporous hydrophobic cloth coated with anti-Salmonella antibody. Using this method, the detection of as few as 10 Salmonella cells per gram of chicken meat was possible within a total of 18 h, which included 16 h of enrichment in either tetrathionate, selenite cystine, or nutrient broths.

Use of polymyxin-coated polyester cloth in the enzyme immunoassay of Salmonella lipopolysaccharide antigens.

Polyester cloth coated with polymyxin B was used to capture Salmonella typhimurium lipopolysaccharide antigens which were then quantitatively or qualitatively assayed using a specific antibody-peroxidase conjugate. This simple, rapid method can be used to assay a large number of samples by employing a large sheet of the polymyxin-coated cloth onto which multiple samples can be blotted. The method is reproducible and economical, since polymyxin B is relatively inexpensive, stable and available in pure form.

Application of polymyxin-coated polyester cloth to the semi-quantitation of Salmonella in processed foods.

A rapid and economical semi-quantitative test for Salmonella cells in foods is proposed. Food samples containing different levels of Salmonella cells were homogenized and serially diluted in enrichment broths and then incubated for about 20 h at 37 degrees C. The presence of Salmonella cells in each dilution was assayed by capturing deoxycholate-extracted Salmonella lipopolysaccharides on a sheet of polymyxin-coated polyester cloth, followed by colorimetric detection with an anti-Salmonella antibody-enzyme conjugate. The minimum dilutions which resulted in no detectable growth were correlated with the extent of Salmonella contamination in the food samples.

Use of detergents in the preparation of Salmonella samples for enzyme immunoassay on polymyxin-coated polyester cloth.

Heating Salmonella cells in the presence of detergents such as deoxycholate or Triton x-100 greatly increased the sensitivity of an enzyme immunoassay for lipopolysaccharide antigens using polymyxin-coated polyester cloth as the solid phase for antigen capture. The presence of a 100-fold excess of E. coli lipopolysaccharide in the test sample did not affect the detection of the Salmonella lipopolysaccharide. The sensitivity of the assay using deoxycholate-heat treatment of the antigens followed by detection on polymyxin-coated cloth was equivalent to that of an assay using antibody-coated polyester cloth for antigen capture.

Rapid and economical detection of Salmonella enteritidis in eggs by the polymyxin-cloth enzyme immunoassay.

A rapid, simple and economical procedure for the detection of Salmonella enteritidis in eggs was developed. The contents of whole eggs inoculated with low numbers of S. enteritidis were mixed with a minimal volume of a nutrient-rich broth (1:2 ratio of egg to broth) and incubated overnight. The lipopolysaccharide (LPS) antigens of S. enteritidis were extracted by heating in the presence of cholate. The antigens were captured on polymyxin-coated polyester cloth, and the captured antigens were detected by sequential reactions with anti-serogroup D1 rabbit antiserum, anti-rabbit antibody-peroxidase conjugate and tetramethylbenzidine substrate solution. This polymyxin-cloth enzyme immunoassay (polymyxin-CEIA) was highly specific for salmonellae bearing the factor O:9 antigen, reacting in the assay of 19 S. enteritidis strains tested, including two rough isolates, but not with salmonellae lacking the factor O:9 antigen or non-Salmonella bacteria. The threshold sensitivity of the polymyxin-CEIA for S. enteritidis suspensions was ca. 10(6) cfu/ml. This combined enrichment culture and polymyxin-CEIA required less than 24 h to complete and detected as few as 1-2 S. enteritidis cfu inoculated into a whole egg. This procedure should facilitate the routine monitoring of S. enteritidis in large numbers of egg samples.

Use of inexpensive O antisera as the detecting antibodies for Salmonella antigens in the polymyxin-cloth enzyme immunoassay.

The application of O antigen-specific antisera to the detection of Salmonella lipopolysaccharide (LPS) antigens was examined in an enzyme immunoassay using polymyxin-coated polyester cloth. The LPS antigens were extracted with deoxycholate and captured on polymyxin-cloth. A mixture of rabbit antisera to Salmonella O antigens was allowed to react with the captured antigens, and the reacted antibodies were detected by an anti-rabbit IgG-peroxidase conjugate. The assay gave positive results with 40 different Salmonella serotypes which represented more than 99.9% of the serogroups isolated from over 122,000 food, feed and environmental samples analysed at Laboratory Services Division, Agriculture Canada, from 1975 to 1990. Strong cross-reactivity with Staphylococcus aureus was eliminated by pregrowth of the organisms in the presence of sodium deoxycholate. The O antisera were commercially available and are more economical than monoclonal or affinity purified polyclonal antibodies.

Effect of temperature and agitation on enrichment of Escherichia coli O157:H7 in ground beef using modified EC broth with novobiocin.

The effects of temperature and agitation on the enrichment of Escherichia coli O157:H7 in meat using modified EC broth with novobiocin (mEC + n) were studied. Enrichment at 37 degrees C was compared to 42 degrees C, both with and without shaking. Incubation at 42 degrees C without shaking effectively suppressed ground beef microflora while allowing good growth of E. coli O157:H7 cells. Cells inoculated into ground meats (beef, pork, turkey) were readily detected by enrichment for 24 h in mEC + n at 42 degrees C without shaking, followed by screening the enrichment cultures using a rapid and inexpensive commercially available enzyme immunoassay system, the E. coli O157 Rapitest.

Salmonella detection by the polymyxin-cloth enzyme immunoassay using polyclonal and monoclonal detector antibodies.

Several commercially available O-antigen polyclonal antisera and a monoclonal antibody to the core region of lipopolysaccharide (LPS) were examined as sources of detector antibodies in a polymyxin-cloth enzyme immunoassay (polymyxin-CEIA) for Salmonella. In this assay, polymyxin-coated polyester cloth captured the LPS antigens from Salmonella broth cultures, followed by immunoenzymatic detection of the captured LPS using specific antibodies. Pools of polyvalent antisera reacted with all of the Salmonella strains tested, but also gave cross-reactions with some non-Salmonella bacteria. On the other hand, the monoclonal antibody gave positive reactions with all of the Salmonella tested except serogroup O-strains, but did not react with any of the non-Salmonella bacteria. The monoclonal antibody supplemented with a single factor serogroup O:35 rabbit antiserum was able to detect the serogroup O-strains without causing any cross-reactions with the non-Salmonella bacteria. As an example of the applicability of this assay system, low levels of Salmonella cells spiked into various food samples were successfully detected after an overnight enrichment in broth.

Assay of Salmonella in enrichment cultures of foods, feeds and environmental samples by the polymyxin-cloth enzyme immunoassay.

A variety of foods, animal feeds and environmental samples were analyzed for the presence of Salmonella using the polymyxin-cloth enzyme immunoassay (p-CEIA) system. Salmonella lipopolysaccharide (LPS) antigens were captured from test samples on polymyxin-coated polyester cloth, followed by immunoenzymatic detection of bound antigens using a monoclonal antibody recognizing an LPS common core oligosaccharide. Dairy and egg products, animal feeds and environmental samples from food processing plants were pre-enriched for 24 h, followed by selective enrichment for a further 24 h in either tetrathionate brilliant green (TBG), selenite cystine (SC) or brain-heart infusion broth containing 0.5% yeast extract, 0.5% cholate and 0.3% selenite (BYCS). The samples were assayed by the p-CEIA after each stage of enrichment. After selective enrichment, the p-CEIA gave results which were in complete agreement with the standard culture technique in the analysis of all foods examined. On the other hand, a combination of selective enrichment and the p-CEIA out-performed the Modified Semi-Solid Rappaport Vassiliadis (MSRV) method in screening pre-enrichment cultures of feeds and environmental samples. Use of the new selective medium BYCS prior to performing the p-CEIA gave the highest recovery of Salmonella from feeds and environmental samples.

Use of hydrophobic cloths as antibody adsorbents for enzyme immunoassay: detection of Brucella antigens.

A cloth-ELISA (C-ELISA) antigen capture assay for the detection of Brucella melitensis and B. abortus was developed. Segments (6-mm squares) of hydrophobic polyester cloth coated with diluted serum from a B. abortus-infected cow were incubated with saline suspensions of heat-killed Brucella cells, or with cultures of bovine or sheep blood or bovine tissue homogenates that had been inoculated with B. abortus or B. melitensis added to trypticase soy broth (TSB) and incubated for 2-3 days. The captured antigen was detected by a bovine anti-Brucella antibody-horseradish peroxidase conjugate system. The enrichment culture technique detected as few as three Brucella colony-forming units (c.f.u.) in 0.5 ml of bovine blood and was positive in cultures in which the Brucella concentration had reached 3 X 10(6) c.f.u. ml-1 (after 2 or 3 days incubation). The combined enrichment-cloth-ELISA method gave complete correlation with cultural isolation and results were available 3 days before colonies appeared in conventional culture. Hydrophobic cloths have potential use in diagnostic procedures since they provide simple, rapid and economical assays.

Swab-based enzyme immunoassay system for detection of meat residues on food contact surfaces as a hygiene monitoring tool.

An enzyme immunoassay system based on the use of a macroporous swab as a solid phase for the capture and subsequent immunoenzymatic detection of immunoglobulin G (IgG) from meat residues on food contact surfaces was developed as a hygiene-monitoring tool. Moistened polyester swabs coated with anti-bovine or anti-chicken IgG were rubbed on the test surface, and the captured IgG was subsequently detected directly on the swabs by brief sequential reactions with anti-bovine or anti-chicken IgG-peroxidase conjugate and chromogenic peroxidase substrate.

Detection of hazelnut proteins in foods by enzyme immunoassay using egg yolk antibodies.

An enzyme immunoassay (EIA) was developed for the detection of hazelnut proteins in foods. This assay used inexpensive chicken egg yolk antibodies in a sandwich EIA format for the immunospecific capture and detection of hazelnut proteins present in a variety of different food matrices. The assay was able to detect less than 1 ppm of hazelnut protein in most of the foods tested and did not exhibit any appreciable cross-reactivity with other nuts or food matrices. This assay will be a useful tool for the food industry and regulatory agencies that wish to test foods for the presence of undeclared hazelnut allergens.

Performance of the Dynabeads anti-Salmonella system in the detection of Salmonella species in foods, animal feeds, and environmental samples.

The immunomagnetic separation Dynabeads anti-Salmonella technique was evaluated for the detection of Salmonella species in a variety of foods, feeds, and environmental samples. Salmonella cells in preenrichment broths were captured using the Dynabeads anti-Salmonella system and were then washed and plated on indicator media. A total of 308 naturally contaminated samples were analyzed, including 46 cheese and egg products, 183 animal feeds, and 79 environmental swabs. The results of the Dynabeads method gave 100% correlation with the results of the standard culture technique used for foods and the modified semisolid Rappaport-Vassiliadis method used for feeds and environmental samples.

Polymacron enzyme immunoassay system for detection of naturally contaminating Salmonella in foods, feeds, and environmental samples.

A simple dot blot enzyme immunoassay was developed to screen enrichment broth cultures for the presence of Salmonella. This unique system utilizes macroporous polyester cloth (Polymacron) with an inexpensive hemoglobin coating to provide a high-affinity adsorbent for lipopolysaccharide (LPS) antigens in test samples. Bound LPS antigens are then detected using a monoclonal antibody conjugate recognizing a core oligosaccharide epitope common to all salmonellae frequently found in foods and related samples. The entire test (not including enrichment culture) could be completed in less than 1 h. The performance of this assay was evaluated in the analysis of enrichment broth cultures from a variety of egg and dairy products, chicken carcasses, animal feeds, and food-processing plant environmental samples for the presence of Salmonella.

Transcriptional enhancement of the Listeria monocytogenes PCR and simple immunoenzymatic assay of the product using anti-RNA:DNA antibodies.

A method was developed to enhance the sensitivity of a Listeria monocytogenes PCR detection system by in vitro transcription of amplicons incorporating bacteriophage T7 RNA polymerase promoter sequences in one of the priming oligonucleotides. The resulting transcript can be detected by hybridization with a DNA probe immobilized in the wells of a microtiter plate, followed by immunoenzymatic assay of the RNA-DNA hybrids with an anti-RNA-DNA hybrid antibody. This highly sensitive method was reactive in the assay of various L. monocytogenes isolates but not with other Listeria or non-Listeria species.

A simple RNA probe system for analysis of Listeria monocytogenes polymerase chain reaction products.

The synthesis of an RNA probe specific for the hlyA gene of Listeria monocytogenes by in vitro transcription from a polymerase chain reaction (PCR)-generated template incorporating bacteriophage T7 promoter sequences is described. This simple method produced a high yield of RNA which hybridized specifically with hlyA PCR products on a membrane, resulting in RNA-DNA hybrids which were detected by an immunoenzymatic assay with an anti-RNA-DNA hybrid antibody. The RNA probe hybridization system was more sensitive in the analysis of the PCR products than was the conventional agarose gel electrophoresis method. When applied to the analysis of PCR samples from cultures of various Listeria and non-Listeria organisms, the RNA probe was reactive in the assay of 62 different L. monocytogenes isolates but not other Listeria species. Among the non-Listeria organisms tested, only Enterococcus faecalis gave a weak positive reaction with more than 10(9) cells per ml. This reactivity disappeared at lower cell densities. This strategy for the synthesis and application of RNA probes should facilitate the analysis of PCR products in the detection of L. monocytogenes and possibly other food pathogens.

Rapid enzyme immunoassay of chicken egg yolk IgG.

A simple and rapid enzyme immunoassay for specific antibodies in chicken egg yolk is described. As a model system, the levels of anti-Salmonella IgG in the yolk of eggs obtained from various produce retailers were compared. Polyester cloth coated with Salmonella typhimurium lipopolysaccharide was used to capture specific egg yolk antibodies, which were then detected using an anti-chicken IgG-peroxidase conjugate. This assay, requiring less than 30 min to complete, revealed considerable differences in the relative levels of anti-Salmonella IgG in the egg yolks. Anti-Salmonella IgG levels were generally lower in eggs obtained from large produce retailers than in eggs obtained from a small local farm. This assay may provide a rapid and economical means of monitoring the levels of Salmonella contamination in chicken rearing facilities.

A simple and inexpensive glucose oxidase substrate system for enzyme immunoassay.

A simple and inexpensive glucose oxidase substrate system was developed for enzyme immunoassay. This system is based on the formation of a coloured complex between polyvinyl alcohol or starch and iodine produced from iodide in the presence of hydrogen peroxide generated by the glucose oxidase reaction. The rate of iodine production was enhanced by the supplementation of molybdate. In an enzyme immunoassay for anti-Salmonella antibodies using glucose oxidase as the indicator enzyme, the molybdate-enhanced polyvinyl alcohol- and starch-glucose-iodide substrate systems were as sensitive as a conventional glucose oxidase assay system employing horseradish peroxidase as a secondary enzyme and a suitable hydrogen-donating chromogen.

Cloth-based hybridization array system for the detection of multiple antibiotic resistance genes in Salmonella enterica subsp. enterica serotype Typhimurium DT104.

AIMS: A simple DNA macroarray system was developed for detection of antibiotic resistance and other marker genes associated with the multidrug-resistant food pathogen Salmonella enterica subsp. enterica serotype Typhimurium DT104. METHODS AND RESULTS: A multiplex polymerase chain reaction (PCR) incorporating digoxigenin-dUTP was used to simultaneously amplify seven marker sequences, with subsequent rapid detection of the amplicons by hybridization with an array of probes immobilized on polyester cloth and immunoenzymatic assay of the bound label. This system provided sensitive detection of the different genetic markers in the S. Typhimurium DT104 genome, giving positive reactions with as few as 10 CFU, and the hybridizations were highly specific, with no reactions of amplicons with heterologous probes on the array. CONCLUSIONS: This cloth-based hybridization array system (CHAS) provides a simple, cost-effective tool for monitoring S. Typhimurium DT104 in foods and their production environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The CHAS is a simple and cost-effective tool for the simultaneous detection of amplicons generated in a multiplex PCR, and the concept is broadly applicable to the detection and characterization of food pathogens.